Elemental Metabolomics in Pregnancy

Abstract

Nutrition is fundamentally important for human health and development; in particular, an adequate supply of macronutrients such as carbohydrates, lipids and proteins as well as specific elements in the form of vitamins and minerals. An insufficient intake of macronutrients or micronutrients leads to poor metabolism and physiological changes associated with a variety of negative health outcomes. Maternal nutrition is perhaps the most important determinant in foetal development in all mammalian species including humans. Additionally, nutrition in pregnancy can have long term effects on the developing infant and has been associated with development of chronic disease later in life. Changes in micronutrient status have been associated with gestational complications such as gestational diabetes mellitus (GDM), preeclampsia (PE), foetal growth restriction (FGR), and preterm birth. Pregnancy disorders such as these are poorly understood despite extensive research, and novel research into this field is desperately required. Recent advancements in technology have allowed the simultaneous measurement of large numbers of elements and metabolites. Currently, we only know how a handful of elements affect pregnancy outcomes, even though many are likely to be essential to human development. This research aimed to determine the elemental nutrition status of pregnant woman and correlate this with (1) weeks gestation, (2) pregnancy health and (3) mechanisms that may contribute to disease, with the ultimate goal of using these values to determine gestational outcomes. This was to be conducted through series of cohort analysis, providing detailed baseline information on micronutrient levels in non-pregnant and pregnant Australians, to then determine if aberrant changes in nutrition are present, and how this may contribute to any disease state.\nIt was critical to establish a means of multi-elemental analysis on ICP-MS that was reproducible and conformed to rigorous quality control standards, ensuring the validity of measurements. This was done through the use of placental cord blood plasma, and in collaboration with an externally accredited laboratory for elemental measurements, ensuring the laboratory facilities and ICP-MS methodologies in place at Griffith University are comparable to a nationally accredited analytical facility. For post method validation, elements within a healthy population had to be determined, to then discern what can be considered abnormal. Elemental reference ranges were established in healthy non-pregnant donors attending the Red Cross Blood Bank (Queensland Red Cross Blood Service). Generally, the levels of elements were comparable to other countries and within published reference ranges. Age was associated with increases in Fe, Se, Cd, Cs, and Pb; whilst Cu, I, and Tl decreased. Sex specific differences included higher levels of Mg, K, Fe, Zn, Se, Rb, Cs, and Pb in males; whilst females had higher concentrations of Co, Cu, As, and Cd. What was interesting, was the varied concentrations of some elements between some regions within the same country, a trend that would be repeated across different studies in this thesis. Having measured the concentrations of elements in a non-pregnant population, it was essential to quantify elements in pregnant women across gestation for comparison and to determine what may be considered normal for gestation. Due to the constant growth and change that is associated with pregnancy, non-pregnant, 18-, 28-, and 36-week pregnant women were tested. There were no signs of elemental deficiencies, or over nutrition in either the non-pregnant or pregnant samples in these studies; nor was there any evidence of pollutants, contaminants, or abnormal levels of heavy metals. It was established that the changes that occur across gestation are extensive, with concentrations varying throughout gestation. However, it appears that there are a number of key factors that may influence these gestational concentrations that need to be considered, namely environmental exposures and collection methods. Further this project then highlighted changes of elements that occurred across gestation, and in pregnancy outcomes such as PE, GDM, FGR, and preterm birth. It assessed the use of elements as a means of predicting gestational outcomes at 18 and 36-weeks, indicating that earlier gestation measurements may provide a better means of discrimination, though validation is required in order to confirm these results. This thesis also evaluated small molecule metabolites and their ability to characterise and differentiate, non-pregnant, healthy pregnant, and poor gestational outcomes, noting significantly better results than elemental analysis. Specifically, in discrimination capabilities with data analysis models, small molecule metabolites outperformed the models constructed based on elements. Although further validation is required before these methods could be applied clinically. Knowledge of the influence of region, collections methods, and time of sample collection were also highlighted through cohorts from across Australia. This thesis proposes the use of a standard operating procedure for elemental and metabolic analysis to ensure the consistency of measurements, aiming to minimise preventable variance in the future.